Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy
Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy
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使用表面等离子共振法和圆二色光谱法评估组织蛋白酶 B 降解 IV 型胶原蛋白的活性
DOI：
10.1016/j.jpba.2014.02.009
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
M. Sugawara
中科院分区：
文献类型：
--
作者：
A. Shoji;M. Kabeya;Y. Ishida;A. Yanagida;Y. Shibusawa;M. Sugawara
用表面等离子体共振(SPR)和圆二色谱(CD)测定了组织蛋白酶B降解IV型胶原和热变性IV型胶原(明胶)的活性。组织蛋白酶B降解各底物的最适pH为4.0左右。ΔRU(15min)是注射组织蛋白酶B后15min SPR信号的下降，由于存在三螺旋构象，IV型胶原的RU小于热变性IV型胶原的RU。CD研究表明，在pH为4.0时，IV型胶原蛋白的三螺旋构象具有不稳定性。由于非螺旋区的蛋白水解性裂解引起的三螺旋构象的局部解离，促进了组织蛋白酶B对IV型胶原的降解。在pH值为4.0时，组织蛋白酶B降解IV型胶原的初始速度与浓度呈双相关系，表明组织蛋白酶B在低浓度时表现出外肽酶活性，而在高浓度时表现出内肽酶活性。从酶的作用机理出发，对组织蛋白酶B对IV型胶原和热处理IV型胶原的外肽酶活性的动力学参数进行了评价和讨论。
Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15 min), which is a decrease in the SPR signal at 15 min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.